Bottom Line:
A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine.PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not.We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes.

ABSTRACTA dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.

Fig7: PDZK1 knockdown (PDZK1 KD) was established in Caco-2BBe/hNHE3V (C2N3) cells. PDZK1 KD cells display significantly reduced acid-activated NHE3 activity compared to control cells. a Total cell lysates from C2N3 cells infected with empty vector lentivirus (control/CTRL) and PDZK1 shRNAs (Sh1 and Sh1 + 3 + 4) were analyzed by Western blots. b Protein bands were quantified using image J software and the values were normalized against β-actin, n = 3. c Immunofluorescence images obtained by confocal microscopy in XZY plane showed a more diffused expression of NHE3-VSV-G in the cytoplasm of PDZK1 KD cells compared to control cells (arrow pointing to NHE3 staining); AP apical membrane, BL basolateral membrane. ***P < 0.0005. d Acid suicide was applied to improve the homogeneity and expression of NHE3 in both control and PDZK1 KD cells, after which a similar pattern of NHE3 expression was obtained in both control and PDZK1 KD cells. Those cells were then used for the NHE3 activity measurements. Scale bar represents 10 μM. e NHE3 activity was measured fluorometrically in C2N3/PDZK1 KD cells, which showed a significant reduction in NHE3 activity compared to control cells. Bar graph represents the results from three to five different passages and includes a total of at least nine cover slips (five regions of interest assessed in each cover slip) for each condition. Bar graphs are represented as mean ± SEM. ***P < 0.0005 compared to controls

Mentions:
A human NHE3 overexpressing colonic cell line Caco-2BBe/hNHE3V was established and PDZK1 expression was >70 % knocked down (Fig. 7a, b). Immediately after the PDZK1 knockdown, the cells displayed mislocalization of NHE3 to the intracellular and basolateral pool, and NHE3 expression was not present in all cells (Fig. 7c). To achieve more homogenous NHE3 expression levels, repeated cycles of “acid suicide selection” were performed both in the control and knockdown (KD) cells. When immunocytochemical analysis of the acid-selected cells revealed similar expression of NHE3 in the apical pole of the Caco-2BBe/hNHE3V empty vector control and the PDZK1 KD cells, they were used for NHE3 transport activity measurements (Fig. 7d). Despite robust apical NHE3 expression, the acid-activated NHE3 activity was significantly lower in PDZK1 KD cells than controls (Fig. 7e).Fig. 7

Fig7: PDZK1 knockdown (PDZK1 KD) was established in Caco-2BBe/hNHE3V (C2N3) cells. PDZK1 KD cells display significantly reduced acid-activated NHE3 activity compared to control cells. a Total cell lysates from C2N3 cells infected with empty vector lentivirus (control/CTRL) and PDZK1 shRNAs (Sh1 and Sh1 + 3 + 4) were analyzed by Western blots. b Protein bands were quantified using image J software and the values were normalized against β-actin, n = 3. c Immunofluorescence images obtained by confocal microscopy in XZY plane showed a more diffused expression of NHE3-VSV-G in the cytoplasm of PDZK1 KD cells compared to control cells (arrow pointing to NHE3 staining); AP apical membrane, BL basolateral membrane. ***P < 0.0005. d Acid suicide was applied to improve the homogeneity and expression of NHE3 in both control and PDZK1 KD cells, after which a similar pattern of NHE3 expression was obtained in both control and PDZK1 KD cells. Those cells were then used for the NHE3 activity measurements. Scale bar represents 10 μM. e NHE3 activity was measured fluorometrically in C2N3/PDZK1 KD cells, which showed a significant reduction in NHE3 activity compared to control cells. Bar graph represents the results from three to five different passages and includes a total of at least nine cover slips (five regions of interest assessed in each cover slip) for each condition. Bar graphs are represented as mean ± SEM. ***P < 0.0005 compared to controls

Mentions:
A human NHE3 overexpressing colonic cell line Caco-2BBe/hNHE3V was established and PDZK1 expression was >70 % knocked down (Fig. 7a, b). Immediately after the PDZK1 knockdown, the cells displayed mislocalization of NHE3 to the intracellular and basolateral pool, and NHE3 expression was not present in all cells (Fig. 7c). To achieve more homogenous NHE3 expression levels, repeated cycles of “acid suicide selection” were performed both in the control and knockdown (KD) cells. When immunocytochemical analysis of the acid-selected cells revealed similar expression of NHE3 in the apical pole of the Caco-2BBe/hNHE3V empty vector control and the PDZK1 KD cells, they were used for NHE3 transport activity measurements (Fig. 7d). Despite robust apical NHE3 expression, the acid-activated NHE3 activity was significantly lower in PDZK1 KD cells than controls (Fig. 7e).Fig. 7

Bottom Line:
A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine.PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not.We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes.

ABSTRACTA dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.